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Optimized DNA and protein sequence of an antibody to improve quality and yield of bacterially expressed antibody fusion proteins

a technology of bacterially expressed antibodies and protein sequences, applied in the direction of antiinfectives, peptides, drug compositions, etc., can solve the problems of induction of undesired immune responses, adverse events in patients, and dramatically increased production costs, so as to achieve dramatic increases in production costs

Active Publication Date: 2011-02-15
CHEMOTHERAPEUTISCHES FORSCHUNGINSTITUT GEORG SPEYER HAUS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

"The patent text discusses the high standards of purity and homogeneity that therapeutic proteins for human patients must meet to be approved by regulatory authorities. By-products in the preparations of the active compound can cause adverse events in patients, so they must be removed to the extent technically possible, and any remaining by-products must be individually demonstrated to have biological activities or absence. This requirement increases production costs due to the need for sophisticated and expensive purification techniques and additional testing if a particular by-product cannot be removed. The patent text also describes the production of a therapeutic protein called scFv(FRP5)-ETA antibody-toxin, which requires a plasmid called pSW220-5 to express it. The purification of this protein involves removing a by-product that migrates below the main band. However, this by-product cannot be removed using standard protein purification techniques."

Problems solved by technology

By-products contaminating preparations of the active compound may cause adverse events in a patient, such as toxic reactions, and / or the induction of undesired immune responses.
As a consequence, production costs will dramatically increase due to the requirement for sophisticated and expensive purification techniques used to remove such undesired by-products, and / or due to additional testing that is required if a particular by-product cannot be removed.
Using standard protein purification techniques, it has not been possible so far to remove this truncated protein fragment.

Method used

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  • Optimized DNA and protein sequence of an antibody to improve quality and yield of bacterially expressed antibody fusion proteins
  • Optimized DNA and protein sequence of an antibody to improve quality and yield of bacterially expressed antibody fusion proteins

Examples

Experimental program
Comparison scheme
Effect test

example 1

Molecular Cloning of Modified Expression Constructs

Method

Construction of scFv(FRP5)-ETA Expression Vectors

[0071]The plasmid pSW220-5 was described before (7). It contains sequences coding for an N-terminal FLAG tag, a first His6 cluster, the ErbB2-specific scFv(FRP5), a second His6 cluster, and truncated Pseudomonas exotoxin A (residues 252-613 of the wildtype toxin) in a single open reading frame. Plasmid pSES211 (unpublished; provided by TopoTarget Germany AG) contains an open reading frame for scFv(FRP5)-ETA originally derived from pSW220-5 and still including the first N-terminal His6 cluster, but lacking the N-terminal FLAG tag and the internal His6 cluster between scFv(FRP5) and exotoxin A sequences. Plasmid pSES212 was derived by deleting the remaining N-terminal His6 cluster of pSES211. The plasmid was generated by PCR using the oligonucleotide primers 5′NdeI-scFv(FRP5) 5′-CGATTAGCATATGCAGGTACAACTGCAGCAGTCAGGACC-3′ (SEQ ID NO:5) and 3′XbaI-scFv(FRP5) 5′-GCTGCCGCCCTCTAGAGCTTT...

example 2

Expression of scFv(FRP5)-ETA Derivatives in E. coli

Method

Expression of scFv(FRP5)-ETA Derivatives in E. coli, Preparation of Inclusion Bodies, Solubilization and Refolding

[0075]E. coli DH5α were transformed with the expression plasmids pSW220-5, pSES212 or pSES213. One liter expression cultures (LB, 0.5% glucose, 50 μg / ml kanamycin in the case of pSES212 or pSES213, or 100 μg / ml ampicillin in the case of pSW220-5) were grown at 37° C. to an OD600 of 0.8. The cultures were induced by addition of 0.5 mM IPTG for 3 hours. Cells were harvested by centrifugation (7500 g, 10 min), resuspended in PBS, and lysed in a French pressure cell. Inclusion bodies were collected by centrifugation (10000 g, 10 min, 4° C.) and washed by resuspension in washing buffer (2 M urea, 2% Triton X-100, 500 mM NaCl in PBS, pH 8) and subsequent centrifugation (10000 g, 10 min, 4° C.). Purified inclusion bodies were resuspended in solubilization buffer (8 M urea, 500 mM NaCl in PBS, pH 8). After centrifugation,...

example 3

Biological Activity of scFv(FRP5-M92S)-ETA

Method

Cells and Culture Conditions

[0078]Murine renal carcinoma cells stably expressing E. coli β-galactosidase (Renca-lacZ), or β-galactosidase and human ErbB2 (Renca-lacZ / ErbB2) (8) were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2 mM glutamine, 100 units / ml penicillin, 100 μg / ml streptomycin, 0.25 mg / ml Zeocin (Invitrogen, Karlsruhe, Germany), and 0.48 mg / ml G418 (Renca-lacZ / ErbB2).

Cell Viability Assay

[0079]Cells were seeded in 96-well plates at a density of 1.5×104 cells / well in normal growth medium. Different concentrations of scFv(FRP5)-ETA fusion proteins or diluent were added to triplicate samples, and the cells were incubated for 48 h at 37° C. in 5% CO2 and 95% humidified air. An aliquot of 10 μl of 10 mg / ml MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide) (Sigma, Deisenhofen, Germany) in PBS was added to each well, and the cells were incubated for another 3 h. Cells were lysed by t...

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Abstract

Object matter of the invention is an optimized DNA sequence encoding the scFv(FRP5) antibody fragment. This novel sequence prevents the generation of the undesired by-product in the context of an scFv(FRP5)-ETA fusion protein, and possibly also other bacterially expressed scFv(FRP5)-containing fusion proteins. The DNA sequence of the scFv(FRP5) domain of scFv(FRP5)-ETA was modified by exchanging a distinct codon, thereby preventing an otherwise possible internal start of protein translation.

Description

[0001]This application claims the benefit of U.S. Provisional Application Ser. No. 60 / 949,580 filed Jul. 13, 2007, the contents of which are incorporated by reference herein in their entirety.[0002]The antibody-toxin scFv(FRP5)-ETA is a recombinant fusion protein composed of a single-chain antibody fragment derived from the ErbB2-specific antibody FRP5, via gene fusion linked to a truncated fragment of Pseudomonas exotoxin A. High and selective antitumoral activity of scFv(FRP5)-ETA against ErbB2 expressing cancer cells in vitro, in animal models and in cancer patients has been described in detail in the literature. Production of scFv(FRP5)-ETA by bacterial expression in E. coli using current methodology results, in addition to the major product of intact scFv(FRP5)-ETA, also in a truncated scFv(FRP5)-ETA fragment as a by-product. Complete elimination of this undesired fragment using classical protein purification techniques has so far not been achieved.[0003]Object matter of the in...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): A61K39/00
CPCA61K31/7088C07K14/21C07K16/32C07K2317/622C07K2319/00C07K2319/55A61P31/00
Inventor WELS, WINFRIED S.DAELKEN, BENJAMINSCHWARZ, SYLVIA E.
Owner CHEMOTHERAPEUTISCHES FORSCHUNGINSTITUT GEORG SPEYER HAUS
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